Implantable particles for tissue bulking and the treatment of gastroesophageal reflux disease, urinary incontinence, and skin wrinkles

ABSTRACT

The invention encompasses the treatment of urinary incontinence, gastroesophageal reflux disease and the amelioration of skin wrinkles using biocompatible hydrophilic cationic microparticles and a cell adhesion promoter.

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional of application Ser. No.09/263,773, filed Mar. 5, 1999, now U.S. Pat. No. 6,335,028, the contentof which is incorporated by reference.

FIELD OF INVENTION

[0003] The present invention relates to tissue bulking, the treatmentgastroesophageal reflux disease, urinary incontinence and theamelioration of skin wrinkles.

BACKGROUND OF INVENTION Gastroesophageal Reflux Disease (“GERD”)

[0004] Although gastroesophageal reflux is a normal physiologicalphenomenon, in some cases it is a pathophysiological situation that canresult in a variety of symptoms which may become severe in extremecases. Gastro-Esophageal Reflux Disease (“GERD”), describes a backflowof acidic and enzymatic liquid from the stomach to the esophagus. Itcauses burning sensations behind the sternum that may be accompanied byregurgitation of gastric acid into the mouth or even the lung.Complications of GERD which define the severity of the disease includeesophageal tissue erosion, and esophageal ulcer wherein normalepithelium is replaced by a pathological tissue.

[0005] Statistical data indicate that about 35% of the Americanpopulation suffer from heartburn at least once a month and between 5 to10% once a day. More importantly for this kind of disease about 2% ofthe American population suffer from GERD based on medical evidence datafrom endoscopic examination. This disease is related to the age ofindividuals and seems to increase after 40 years of age. (Nebel O. T. etal., Am. J. Dig. Dis., 21(11) :953-956 (1976)).

[0006] In normal patients, after a meal the lower esophageal sphincterremains closed, but in patients with GERD, it relaxes and allows someacidic material to reflux into the esophageal tube as a result ofstomach contractions. Actually GERD can be attributed primarily totransient relaxation of the lower esophageal sphincter. In other cases,GERD can be attributed to decreased resting tone of the lower esophagealsphincter or to congenital small dimension of the sphincter itself.Other causes also exist which contribute to varying degrees to theexistence and severity of this disease.

[0007] In addition, there are external factors that contribute toexacerbate the symptoms of GERD, which conditions include eating fattyfoods, caffeine intake, smoking, tight clothing and certain medications.Decrease in salivation can also be a factor that exacerbates GERD, sinceunder normal conditions saliva, which is an alkaline liquid, aids inneutralizing acidic reflux and therefore diminishing the duration of theacidic exposure of the esophagus.

[0008] Erythema is one of the first visible signs of GERD, which can beseen by endoscopy. Tissue erosion indicates more advanced disease whichcan then become deep ulcers and lead to cancer (adenocarcinoma increasesin incidence faster than other types of cancer). Diffuse ulceration andspecific complications occur in about 3.5% of patients less than 65years of age with esophageal obstruction, blood loss, and in some cases,perforation. Ulcerative situations not only lead to complications, butthey are also more resistant to treatments. Although severecomplications are uncommon in young patients, they occur in about 20-30%of patients over 65 (Reynolds J. C, Am. J. Health-Sys. Pharm 53,(1996)).

[0009] Prior to the present invention, in an attempt to increase thefunction of the sphincter, bulking methods using bovine collagen andTeflon paste have been used in patients. Both methods have beenunsuccessful, however, as these materials migrate over time from theinitial site of implantation.

[0010] At present, GERD is generally managed by over-the-counter (“OTC”)antacids or prescription drugs, including proton pump inhibitors,motility agents and H₂ blockers. In addition, a portion of GERD patientsrequire surgical intervention; the most common type of surgery isfundoplication which can be done by conventional surgical techniques, orusing laparoscopic techniques. However, fundoplication surgery carriesthe risk of serious side effects and is only marginally successful incuring GERD. Respiratory symptoms are also associated with GERD in about50% of patients, and in patients undergoing fundoplication, theserespiratory symptoms can even increase (76% reported in a study byJohnson W. E. et al., Archives of Surgery, 131:489-492 (1996)).

Urinary Incontinence

[0011] Urinary incontinence is a prevalent problem that affects peopleof all ages and levels of physical health, both in the community atlarge and in healthcare settings. Medically, urinary incontinencepredisposes a patient to urinary tract infections, pressure ulcers,perineal rashes, and urosepsis. Socially and psychologically, urinaryincontinence is associated with embarrassment, social stigmatization,depression, and especially for the elderly, an increased risk ofinstitutionalization (Herzo et al., Ann. Rev. Gerontol. Geriatrics, 9:74 (1989)). Economically, the costs are astounding; in the United Statesalone, over ten billion dollars per year is spent managing incontinence.

[0012] Incontinence can be attributed to genuine urinary stress (urethrahypermobility), to intrinsic sphincter deficiency (“ISD”), or both. Itis especially prevalent in women, and to a lesser extent incontinence ispresent in children (in particular, ISD), and in men following radicalprostatectomy.

[0013] One approach for treatment of urinary incontinence involvesadministration of drugs with bladder relaxant properties, withanticholinergic medications representing the mainstay of such drugs. Forexample, anticholinergics such as propantheline bromide, and combinationsmooth muscle relaxant/anticholinergics such as racemic oxybutynin anddicyclomin, have been used to treat urge incontinence. (See, e.g., A. J.Wein, Urol. Clin. N. Am., 22:557 (1995)). Often, however, such drugtherapies do not achieve complete success with all classes ofincontinent patients, and often results in the patient experiencingsignificant side effects.

[0014] Besides drug therapies, other options used by the skilled artisanprior to the present invention include the use of artificial sphincters(Lima S. V. C. et al., J. Urology, 156:622-624 (1996), Levesque P. E. etal., J. Urology, 156:625-628 (1996)), bladder neck support prosthesis(Kondo A. et al., J. Urology, 157:824-827 (1996)), injection ofcrosslinked collagen (Berman C. J. et al., J. Urology, 157:122-124(1997), Perez L. M. et al., J. Urology, 156:633-636 (1996); Leonard M.P. et al., J. Urology, 156:637-640 (1996)), and injection ofpolytetrafluoroethylene (Perez L. M. et al., J. Urology, 156:633-636(1996)).

[0015] A recent well known approach for the treatment of urinaryincontinence associated with ISD is to subject the patient toperiurethral endoscopic collagen injections. This augments the bladdermuscle in an effort to reduce the likelihood of bladder leakage orstress incontinence.

[0016] Existing solutions to circumvent incontinence have well knowndrawbacks. The use of artificial sphincters for children withintractable incontinence requires long term surveillance of the urinarytract because of the potential for renal failure after device placement(Levesque P. E. et al., J. Urology, 156:625-628 (1996)). Whileendoscopically directed injections of collagen around the bladder neckhas a quite high success rate in sphincter deficiency with nosignificant morbidity, the use of collagen can result in failures thatoccur after an average of two years and considerations need to be givento its cost effectiveness (Khullar V. et al., British J. Obstetrics &Gynecology, 104:96-99 (1996)). In addition, deterioration of patientcontinency, probably due to the migration phenomena (Perez L. M. et al.)may require repeated injections in order to restore continency(Herschorn S. et al., J. Urology, 156:1305-1309 (1996)).

[0017] The results with using collagen following radical prostatectomyfor the treatment of stress urinary incontinence have also beengenerally disappointing (Klutke C. G. et al., J. Urology, 156:1703-1706(1996)). Moreover, one study provides evidence that the injection ofbovine dermal collagen produced specific antibodies of IgG and IgAclass. (McCell and, M. and Delustro, F., J. Urology 155, 2068-2073(1996)). Thus, possible patient sensitization to the collagen could beexpected over the time.

[0018] Despite of the limited success rate, transurethral collageninjection therapy remains an acceptable treatment for intrinsicsphincter deficiency, due to the lack other suitable alternatives.

Skin Wrinkles

[0019] Damage to the skin due to aging or exposure to the sun and otherelements often results in wrinkles and other skin anomalies. In order toremove wrinkles from the skin, people often resort to cosmetic surgery,such as face lifts and skin tucks. In addition, collagen injections havebeen used to remove or ameliorate skin wrinkles. Collagen injectionshave also been used for tissue bulking or to increase the fullness ofcertain body parts, e.g., to increase the fullness of lips or around theeyes and eyebrow area of the face. However, collagen is a naturallyoccurring substance which the body may enzymatically degrade andeliminate over time, thus requiring repeat treatments. Even morealarming from a cosmetic perspective, collagen may move from the initialsite of injection, causing unsightly bumps and bulges under the skin atundesired locations.

[0020] Microbeads or solid microparticles have been used for thecorrection of skin wrinkles. For examples, silicone particles, TEFLONpaste, collagen beads and polyacrylic microspheres have been used withdisappointing results due to, inter alia, adverse tissue reactions,biological degradation and migration from the initial implantationlocation.

MicroParticles

[0021] Prior to the present invention, microspheres have beenmanufactured and marketed for in vitro use in anchorage dependent cellculture. (Van Vezel, A. L., Nature, 216:64-65 (1967); Levine et al.,Somatic Cell Genetics, 3:149-155 (1977); Obrenovitch et al., Biol.Cell., 46:249-256 (1983)). They have also been used in vivo to occludeblood vessels in the treatment of arteriovascular malformation, fistulasand tumors (See, U.S. Pat. No. 5,635,215, issued Jun. 3, 1997 toBoschetti et al.; Laurent et al., J. Am. Soc. Neuroiol, 17:533-540(1996); and Beaujeux et al. J. Am. Soc. Neuroial, A:533-540 (1996)).

[0022] Further, direct implantation of cells into living tissues such asbrain or liver to correct specific deficiencies has been attemptedalbeit with a number of failures. The major problems associated withdirect cell transplantation are the long term viability of the cellransplant and the immunopathological as well as histological responses.Microparticles with cells attached on their surface have been used insome in vivo applications. Cherkesey et al., IBRO, 657-664 (1996),described the culture of adrenal cells on coated dextran beads and theimplantation into mammalian brain to supplant some specific disordersrelated to 6-hydroxydopamine-induced unilateral lesions of thesubstantia nigra. The pre-attachment of cells to dextran microcarriersallowed for improved functions of the cells implanted into the brain.Also liver cells transplantation has been used to manage acute liverfailure, or for the replacement of specific deficient functions such asconjugation of bilirubin or synthesis of albumin. For this purpose, anintrasplenic injection of hepatocytes grown on the surface ofmicrospheres was performed (Roy Chowdhury et al., in: Advanced Researchon Animal Cell Technology, AOA Miller ed., 315-327, Kluers Acad. Press,1989).

[0023] Most of cell implant results have been, however, largelydisappointing for the designated functions (or have had low levels ofbiological function).

SUMMARY OF INVENTION

[0024] The present invention encompasses the use of implantablemicroparticles in the treatment of GERD, urinary incontinence and skinwrinkles. In each use the particles are implanted into the appropriatetissue, muscle, organ etc. as a bulking agent. Further, in each use themicroparticles are preferably pre-coated, with autologous cells, forexample, muscle cells, fat cells and the like. The microparticles of theinvention are biocompatible non-toxic polymers coated with, linked to orfilled with cell adhesion promoters. The microparticles preferablycontain a positive charge on their surface by way of a cationic monomeror polymer.

[0025] In one embodiment, the invention encompasses the treatment ofgastroesophageal reflux disease in a human which comprises implantinghydrophilic biocompatible microparticles comprising (a) a positivecharge and a cell adhesion promoter; and (b) autologous cells layered onthe surface of said beads, into the lower esophageal sphincter. Themicroparticles are preferably microspheres or microbeads which aredescribed in detail herein. The autologous cells are preferably takenfrom the area where the implantation is to be made. Serum or whole bloodtaken from the patient can be used to wash the microparticles prior toimplantation. For GERD treatment implantation may also be made by usingstandard techniques known to the skilled artisan, such as injection (orinjections) via syringe or other suitable devices.

[0026] In yet another embodiment, the invention encompasses thetreatment of urinary incontinence in a human which comprises implantinghydrophilic biocompatible microparticles comprising (a) a positivecharge and a cell adhesion promoter; and (b) autologous cells layered onthe surface of the beads, into the urinary sphincter. The microparticlesare preferably microspheres or microbeads as described herein. Further,the autologous cells are preferably taken from the area where theimplantation is to be made. Serum or whole blood from the patient can beused to wash the microparticles prior to implantation. Implantation isgenerally made using a syringe or other device suitable for theparticular tissue of implantation.

[0027] In another embodiment, the invention encompasses a method oftreating skin wrinkles in a human which comprises the administration orimplantation of microparticles comprising a hydrophilic copolymer havinga positive charge, and a cell adhesion promoter, which microparticleshave been pre-treated with autologous cells. The microparticles can besimply exposed to the autologous cells or mixed thoroughly withautologous cells prior to implantation.

[0028] It should be recognized that both treatments for GERD and urinaryincontinence described above can be used in combination withconventional therapies now used to treat these diseases i.e., oraldiuretics, antacids, suitable drug therapy and the like. Suchcombination therapy can lead to a faster, safer and more comfortablerecovery for the patient.

[0029] In yet another embodiment, the invention encompasses thetreatment or amelioration of skin wrinkles which comprises administeringhydrophilic biocompatible microparticles comprising: (a) a positivecharge and a cell adhesion promoter; and (b) autologous cells, collagen,collagen derivatives or glucosaminoglycans layered on the surface of thebeads, into the area of or surrounding the skin wrinkles. In otherwords, microspheres or microbeads coated with a cell adhesion promoterand pre-treated with the appropriate tissue bulking cells, areadministered to the area of treatment.

[0030] As used herein the terms “administered”, “implanted”, or“implantation” are used interchangeably and mean that the material isdelivered to the area of treatment by techniques know to those skilledin the art and appropriate for the disease to be treated. Both invasiveand non-invasive methods may be used for delivery.

BRIEF DESCRIPTION OF THE DRRAWINGS

[0031]FIG. 1 is a schematic representation of sphincter bulking. Beadsare coated and injected under physiological conditions into thesphincter. The sphincter volume increases proportionally to the amountof injected beads and the lumen size decreases. The beads areprogressively and non-reversibly integrated within the muscles.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention uses microparticles, particularlymicrobeads, having a positive charge on its surface and a cell adhesionpromoter and optionally, a cell growth promoting agent, to treat GERD,urinary incontinence, and skin wrinkles. The microparticles of theinvention are preferably used with autologous cells. In other words, themicroparticles of the invention are colonized with the appropriate cellsprior to implantation. This pre-implantation step has been shown toreduce or eliminate immunological responses and implantation rejectionreactions. Further, the use of non-biodegradable biologically compatiblemicrobeads with positive charges and autologous cells, whethertissue-specific or not, improves tissue acceptance and overalltreatment.

[0033] According to the methods of the present invention, treatment ofGERD, urinary incontinence, and skin wrinkles is achievable whileavoiding or substantially reducing adverse tissue reactions, includingimplantation rejection, degradation of particles, resorption, migrationand other adverse events. The methods of the invention also involveincreased connective tissue response.

[0034] Microbeads or microparticles for use in the present invention arebased on a biocompatible non-toxic polymer coated with agents whichpromote cell adhesion. Living cells attach to the microparticles forminglayered cells therein which link with surrounding tissues to enhancelong term stability of the beads.

[0035] Microparticles intended to be implanted in various locations ofthe body according to the present invention are composed of anon-resorbable hydrophilic polymer containing the appropriate materialfor cell adhesion, and may additionally contain radiopaque molecules orother marking agents, to facilitate localization by radiology prior toor during intervention. Hydrophilic copolymers usable for thisapplication are those of the acrylic family such as polyacrylamides andtheir derivatives, polyacrylates and their derivatives as well aspolyallyl and polyvinyl compounds. All of these polymers are crosslinkedso as to be stable and non-resorbable, and can contain within theirstructure other chemicals displaying particular properties, such aschemotactic effects, promotion of cell adhesion to cells or tissues,such as cells of the esophagus wall or the urethra wall, or skin cells,and/or marking agents.

[0036] The microparticles for use in the present invention are non-toxicto tissues and cells, biocompatible, and adhesive to various cells andtissues at the site of implantation by means of the cell growth theypromote. In addition, these microparticles are non-resorbable andnon-biodegradable, and thus are stable, durable, and will maintain theirgeneral shape and position once implanted at a desired site.

[0037] In general, microparticles for use in the present invention mayhave any shape, with microparticles which are spherical in shape beingpreferred. Microparticles for use in the present invention may havediameters ranging between about 10 μm to about 1000 μm. Preferably,microparticles for use in the present invention which have cells adheredto the surface thereof will have diameters ranging between 50 μm and1000 μm.

[0038] Possible variations of the present invention include replacingthe microparticles with any biocompatible, non-toxic non-resorbablepolymeric particles, membrane, fibers or other solid substrates treatedwith an agent promoting cell adhesion. The invention also includeslinear soluble polymers which, after injection, crosslink in situ toconstitute a solid, cell adhesion promoting filling agent. Preparationand/or injection of empty microparticles (microbubbles) that areprepared in advance or are generated in place via the use of appropriatecatheters, are also contemplated in this invention.

[0039] The microparticles, or other solid substrates, for use in thepresent invention are flexible, such that they can asily pass into andthrough injection devices and small catheters without being permanentlyaltered, but the microparticles are also resistant to the musclecontraction stress generated during and after the implantation process.They are also thermally stable which allows for easy, convenientsterilization, and frozen storage.

[0040] The microparticles, or other solid substrates, for use in thepresent invention are also stable in suspension which allows themicroparticles or other solid substrates to be formulated and stored insuspension and injected with different liquids. More specifically, thehydrophilic nature of the microparticles permits placing them insuspension, and in particular, in the form of sterile and pyrogenic(pyrogen-free) injectable solutions, while avoiding the formation ofaggregates or adhesion to the walls of storage containers andimplantation devices, such as catheters, syringes, needles, and thelike. Preferably, these injectable solutions contain microparticles orother solid substrates distributed approximately in caliber segmentsranging between about 10 μm and about 2000 μm.

[0041] The microparticles of the present invention are both hydrophilicand cationic. The microparticles preferably comprise a copolymer of aneutral hydrophilic monomer, a difunctional monomer, one or moremonomers having a cationic charge, and optionally, a functionalizedmonomer capable of rendering the microparticle detectable. Themicroparticles may also comprise one or more cell adhesion promoters anda marking agent.

[0042] The copolymer is preferably a hydrophilic acrylic copolymer whichcomprises in copolymerized form about 25 to about 98% neutralhydrophilic acrylic monomer by weight, about 2 to about 50% difunctionalmonomer by weight and about 0 to about 50% by weight of one or moremonomers having a cationic charge.

[0043] By way of example, the copolymers described in French Patent2,378,808, which is incorporated herein by reference, can be used inaccordance with this invention to prepare the base microparticlecopolymer.

[0044] As hydrophilic acrylic monomer, acrylamide and its derivatives,methacrylamide and its derivatives or hydroxymethylmethacrylate can beused.

[0045] Examples of difunctional monomer, include but are not limited tothe N,N′-methylene-bis-acrylamide, N′,N′-diallyltartiamide orglyoxal-bis-acrylamide.

[0046] Further, the monomer having a cationic charge, includes but isnot limited to those carrying a tertiary or quaternary amine function,preferably diethylaminoethyl acrylamide, methacrylamidopropyltrimethylammonium or acrylamidoethyl triethylammonium.

[0047] In a particularly preferred embodiment, a copolymer comprisingabout 25 to about 98% methacrylamide by weight, about 2 to about 50%N,N-methylene-bis-acrylamide by weight is used.

[0048] In one particularly advantageous embodiment of the invention, itis possible to increase the stability of the microspheres byreticulating the adhesion agent. By way of example, in the case ofgelatin, the reticulating agent can be chosen among the difunctionalchemical agents reacting on the gelatin amines (e.g., glutaraldehyde,formaldehyde, glyoxal, and the like).

[0049] The functionalized monomer is generally obtained by chemicalcoupling of the monomer with a marker, which can be:

[0050] a chemical dye, such as Cibacron Blue or Procion Red HE-3B,making possible a direct visualization of the microspheres (Bosahetti,J. Biochem-Biophys. Meth., 19:21-36 (1989)). Examples of functionalizedmonomer usable for this type of marking N-acryloyl hexamethyleneCibacrone Blue or N-acryloyl hexamethylene Procion Red HE-3B;

[0051] a magnetic resonance imaging agent (erbium, adolinium ormagnetite);

[0052] a contrasting agent, such as barium or iodine salts, (includingfor example acylamino-e-propion-amido)-3-triiodo-2,4,6-benzoic acid,which can be prepared under the onditions described by Boschetti et al.(Bull. Soc. Chim., No. 4 France, (1986)). In the case of barium ormagnetite salts, they can be directly introduced in powered form in theinitial monomer solution.

[0053] As indicated above it is also possible to mark the icrospheresafter their synthesis. This can be done, for example, by grafting offluorescent markers derivatives (including for example fluoresceinisothiocyanate (FITC), rhodamine isothiocyanate (RITC) and the like).

[0054] Various types of cell adhesion promoters well known in the artmay be used in the present invention. In particular, cell adhesionpromoters can be selected from collagen, gelatin, glucosaminoglycans,fibronectins, lectins, polycations (such polylysine, chitosan and thelike), or any other natural or synthetic biological cell adhesion agent.

[0055] Preferably, the cell adhesion promoter is present in themicroparticle, or other solid substrate, in an amount between about 0.1to 1 g per ml of settled microparticles.

[0056] Microparticles are prepared by suspension polymerization,drop-by-drop polymerization or any other method known to the skilledartisan. The mode of microparticle preparation selected will usuallydepend upon the desired characteristics, such as microparticle diameterand chemical composition, for the resulting microparticles. Themicroparticles of the present invention can be made by standard methodsof polymerization described in the art (see, e.g., E. Boschetti,Microspheres for Biochromatography and Biomedical Applications. Part I,Preparation of Microbands In: Microspheres, Microencapsulation andLiposomes, John Wiley & Sons, Arshady R., Ed., 1998 (in press) which isincorporated herein by reference). Microspheres are prepared startingfrom an aqueous solution of monomers containing adhesion agents such ascollagen (gelatin is a denatured collagen). The solution is then mixedwith a non-aqueous-compatible solvent to create a suspension ofdroplets, which are then turned into solid gel by polymerization ofmonomers by means of appropriate catalysts. Microspheres are thencollected by filtration or centrifugation and washed.

[0057] Cell adhesion promoters or marking agents are introduced onmicrobeads by chemical coupling procedures well known in affinitychromatography, referred to by the term “ligand immobilization”. Anothermethod of introduction is by diffusion within the gel network thatconstitutes the bead and then trapping the diffused molecules in placeby precipitation or chemical cross-linking. Therapeutic agents, drugs orany other active molecules that are suitable for transportation by thebeads can also be introduced into the microbeads prior to beadimplantation according to this last method.

[0058] The microspheres of the invention can also be obtained bystandard methods of polymerization described in the art such as FrenchPatent 2,378,808 and U.S. Pat. No. 5,648,100, each of which isincorporated herein by reference. In general, the polymerization ofmonomers in solution is carried out at a temperature ranging betweenabout 0° C. and about 100° C. and between about 40° C. and about 60° C.,in the presence of a polymerization reaction initiator.

[0059] The polymerization initiator is advantageously chosen among theredox systems. Notably, it is possible to use combinations of an alkalimetal persulfate with N,N,N′,N′-tetramethylethylenediamine or withdimethylaminopropionitrile, organic peroxides such as benzoyl peroxidesor even 2,2′-azo-bis-isobutyronitrile.

[0060] The quantity of initiator used is adapted by one skilled in theart to the quantity of monomers and the rate of polymerization sought.

[0061] Polymerization can be carried out in mass or in emulsion.

[0062] In the case of a mass polymerization, the aqueous solutioncontaining the different dissolved constituents and the initiatorundergoes polymerization in an homogeneous medium. This makes itpossible to access a lump of aqueous gel which can then be separatedinto microspheres, by passing, for example, through the mesh of ascreen.

[0063] Emulsion or suspension polymerization is the preferred method ofpreparation, since it makes it possible to access directly microspheresof a desired size. It can be conducted as follows: The aqueous solutioncontaining the different dissolved constituents (e.g., differentmonomers, cell adhesion agent), is mixed by stirring, with a liquidorganic phase which is not miscible in water, and optionally in thepresence of an emulsifier. The rate of stirring is adjusted so as toobtain an aqueous phase emulsion in the organic phase forming drops ofdesired diameter. Polymerization is then started off by addition of theinitiator. It is accompanied by an exothermic reaction and itsdevelopment can then be followed by measuring the temperature of thereaction medium.

[0064] It is possible to use as organic phase vegetable or mineral oils,certain petroleum distillation products, chlorinated hydrocarbons or amixture of these different solutions. Furthermore, when thepolymerization initiator includes several components (redox system), itis possible to add one of them in the aqueous phase beforeemulsification.

[0065] The microspheres thus obtained can then be recovered by cooling,decanting and filtration. They are then separated by size category andwashed to eliminate any trace of secondary product.

[0066] The polymerization stage can be followed by a stage ofreticulation of the cell adhesion agent and possibly by a marking agentstage in the case of microspheres rendered identifiable by graftingafter synthesis.

[0067] Microparticles of the present invention which have the specificproperties of cell adhesion and growth promotion can be used directlyfor tissue bulking. Moreover, the microparticles of the presentinvention can have specific autologous cells grown on their surface invitro, thereby making the microparticles particularly useful for tissuebulking.

[0068] Prior to the present invention, the injection of implantablesubstances suspended in a physiological solution into a tissue resultedin the formation of discrete aggregates inside the muscle mass. Thesediscrete aggregates can constitute various amounts of the implantedsubstance which stays together, however, the substance does not becomeattached to or a part of the tissue itself. This detachment allows theimplanted substance to move from the original implantation site.

[0069] According to the present invention, in order to avoid thisproblem, the microparticles may be injected individually and separately,or more preferably, the surface of the microparticles may be colonizedby a layer of cells for better integration and long term stability ofthe implant.

[0070] Microparticles of the present invention demonstrate superiorability to grow cells on their surfaces. For example, primary musclecells have been successfully adhered to the surface of themicroparticles of the present invention thereby allowing for a betterintegration within a muscle tissue. In addition, since the ultimate goalof tissue bulking is to artificially increase tissue mass, preadipocyteshave also been used to colonize the surface of the microparticles priorinjection. In this case, the preadipocytes have a volume similar to anyother regular cell, but after implantation when the preadipocytes aresubject to in vivo physiological conditions, they accumulate go dropletsof fats thereby increasing the mass of the implant by more than 10% involume.

[0071] According to the present invention, one means of performingtissue bulking in a patient can be described as follows:

[0072] a) Primary cells are extracted from the patient by a simplebiopsy and isolated;

[0073] b) These cells are grown on the surface of the microparticlesunder growth promoting conditions (e.g., possibly using a nutrient mediawhich contains autologous serum (drawn from the patient), untilconfluence);

[0074] c) the microparticles having the patient's cells grown on the topare injected into the patient's target tissue to be bulked.

[0075] For the treatment of GERD, the microparticles, or other solidsubstrates, are introduced via the esophagus, either by endoscopicdelivery or by laparoscopic technique, and are injected into the wallsof the sphincter where the esophagus meets the stomach, i.e., the loweresophageal sphincter. This decreases the internal lumen of the sphinctermuscle thus permitting easier contraction of the muscle with reducedregurgitation of the gastric fluids into the esophagus. In addition,this treatment reduces the inflammation of the lower esophagus. Themicroparticles, or other solid ubstrates, may also be loaded with X-rayopaque dye or other maging agents for subsequent X-ray visualization.

[0076] In another embodiment, microparticles injected into the phincterat the junction of the esophagus and stomach in rder to treat GERD mayalso include an amount of a drug used o treat GERD, such as H₂ histamineantagonists including imetidine, ranitidine, famotidine and nizatidine;inhibitors of H⁺, K⁺-ATPase including omeprazole and lansoprazole;ntacids including e.g., Al(OH)₃, Mg(OH)₂, and CaCO₃. As with hetreatment of urinary incontinence and skin wrinkles, the icrospheres mayalso be used with anti-inflammatory agents, ngiogenesis inhibitors,radioactive elements, and antimitotic agents.

[0077] Other therapeutic agents to be used in combination with themicrospheres or microparticles of the present invention include thosefor the treatment of skin disorders, GERD and urinary incontinence asreported in Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., McGraw-Hill (1996) and The Physicians's DeskReference® 1997.

[0078] The primary advantages of the method of treating GERD accordingto the present invention over the prior art methods are:

[0079] a) Less invasive effects on the patient compared to surgery;

[0080] b) More permanent effects over antacids or other drugs;

[0081] c) Good biocompatibility with chemotactic effects; and

[0082] d) Ability to use X-ray visualization or MRI to assist infollow-up evaluation of the patient.

[0083] For the treatment of urinary incontinence, the microparticles, orother solid substrates, are introduced via the urethra and injected intothe walls of the bladder sphincter, decreasing the internal lumen of thesphincter muscle thus permitting easier contraction of the muscle withreduced likelihood of incontinence. The microparticles, or other solidsubstrate, may also be loaded with X-ray opaque dye, or other imagingagents for subsequent X-ray visualization.

[0084] In another embodiment, microparticles injected into the bladdersphincter in order to treat urinary incontinence may also include anamount of a drug used to treat urinary incontinence, such asantidiuretics, anticholinergics, oxybutynin and vasopressins.

[0085] Injected microparticles can generate some transient adversereactions such as local inflammation, therefore the microparticles cancontain or be injected with anti-inflammatory drugs, such as salicylicacid derivatives including aspirin; para-aminophenol derivativesincluding acetaminophen; non-steroidal anti-inflammatory agentsincluding indomethacin, sulindac, etodolac, tolmetin, diclodfenac,ketorolac, ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen,oxaprozin; anthranilic acids including mefenamic acid, meclofenamicacid; enolic acids such as piroxicam, tenoxicam, phenylbutazone,oxyphenthatrarone; and nabumetone. These anti-inflammatories arepreferably adsorbed on the microparticle's network and released slowlyover a short period of time (a few days).

[0086] The microparticles may also be used to release other specificdrugs which can be incorporated within the microparticle network beforeinjection into the patient. The drug would be released locally at thesite of implantation over a short period of time to improve the overalltreatment.

[0087] Incorporation of active molecules, such as drugs, into themicroparticles of the present invention can be accomplished by mixingdry microparticles with solutions of said active molecules or drugs inan aqueous or hydro-organic solution. The microparticles swell byadsorbing the solutions and incorporate the active molecule of interestinto the microparticle network. The active molecules will remain insidethe microparticle due to an active mechanism of adsorption essentiallybased on ion exchange effect. The microparticles by their nature carrycationic groups and have the ability to adsorb anionic molecules, suchas well known anti-inflammatory drugs, and these anionic molecules arethen released slowly upon injection into the patient due to the actionof physiological salt and pH. The ability of various types ofmicroparticles to adsorb drug molecules may be readily determined by theskilled artisan, and is dependent on the amount of cationic monomerspresent in the initial solution from which the microparticles areprepared.

[0088] Some of the primary advantages of treating urinary incontinenceaccording to the present invention over prior art methods are:

[0089] a) More permanent effect than the use of regular viscoussolutions of collagen;

[0090] b) Good biocompatibility with chemotactic effect;

[0091] c) Visualization under X-ray or MRI to assist in follow-upevaluation; and

[0092] d) Preventing repeated treatments with resorbable naturallyoccurring substances like collagen.

[0093] The primary advantages of the method of treating skin wrinklesaccording to the present invention are:

[0094] (a) less invasive effects on the patient compared to surgery;

[0095] (b) more permanent effects than the use of collagen injections;and

[0096] (c) good biocompatibility with chemotactic effects.

[0097] For treatment of skin wrinkles, the microparticles may beintroduced via injection. The microparticles may also include one ormore anti-inflammatory agents. The invention is further defined byreference to the following examples that describe in detail thepreparation of microparticles for use in tissue bulking, and thetreatment of skin wrinkles, urinary incontinence, and GERD. Thefollowing examples are illustrative only and should in no way limit thescope of the present invention. It will be apparent to those skilled inthe art that many modifications, both to materials and methods, may bepracticed without departing from the purpose and scope of thisinvention.

EXAMPLES Example 1 Preparation of Irregular Hydrogel Particles withChemotactic Properties

[0098] 58 grams of sodium chloride and 27 grams of sodium acetate weredissolved at room temperature in 100 ml of demineralized water. To thissolution 400 ml of glycerol were added, the pH was adjusted to 6.0 andmonomers were then dissolved. More specifically to this solution 90 gramof methylolacrylamide, 2 g ofmethacrylamidopropyl-trimethyl-ammonium-chloride hydrochloride and 10gram of N,N′-methylene-bis-acrylamide were added and the mixture wasagitated until complete solubilization. The solution was heated at about70° C. and 100 ml of a solution of gelatin at a concentration of 500mg/ml was added. The total volume of the mixture was then adjusted to1000 ml by addition of demineralized water. Finally 20 ml of 70 mg/mlammonium persulfate aqueous solution and 4 ml ofN,N,N′,N′-tertamethyl-ethylene-diamine was added. The obtained mixturewas stored at 70° C. for about 3 hours until formation of a compactthree-dimensional gel. This gel was totally insoluble in water. It wascut in small pieces and then ground to get very small particles of adimension close to 100-200 μm. The particles were then suspended in 1liter of physiological buffer containing 5% (w/v) glutaraldehyde andwere shaken for two hours. Finally the particles were extensively washedto eliminate unpolymerized material, byproducts and salts. To obtainhomogeneous particle size distribution the particle suspension wassieved using an appropriate sieving net.

[0099] These particles possess the characteristics desired for tissuecell adhesion prior to muscle bulking and include cationic groups andadhesion agents for an effective cell adhesion mechanism.

Example 2 Preparation of Spherical Polyacrylic Hydrofel Gel Particleswith Chemotactic Properties

[0100] The solution of monomers prepared as described in Example 1 abovewas poured slowly into 1500 ml of stirred and hot paraffin oil (50-70°C.). After a few minutes a suspension/emulsion of liquids was obtained(the aqueous monomer solution was dispersed into oil and forms verysmall spherical droplets) and the polymerization occurred in suspension.The microdroplets were transformed into microbeads. The solid microbeadswere recovered by centrifugation and suspended in 1 liter ofphysiological buffer containing 5% (w/v) glutaraldehyde and shaken fortwo hours. Finally the particles were extensively washed with water toeliminate completely the oil traces. Organic solvent extraction can beused for a more effective oil removal or an extensive washing in thepresence of traces of nonionic detergents. The obtained microbeads arecalibrated if necessary by sieving through a nylon net and sterilized inan autoclave. These microspheres possess desired characteristics andproperties for cell adhesion prior to muscle bulking.

Example 3 Preparation of Hydrophilic Spherical Polystyrene CopolymerParticles Useful for Tissue Bulking

[0101] 10 gram of styrene is mixed with 60 ml of toluene. 1 gram ofdivinylbenzene, 1 gram of dimethyl-aminoethylmethacrylate and 1 gram ofdimethyl-acrylamide are added to the resulting solution. After completesolubilization the monomer solution is mixed with 1% of AIBN(2,2′-azobisisobutyronitrile) as a polymerization catalyst and with 40ml of paraffin oil as a viscosity inducer agent. The mixture is pouredin an agitated water solution containing 0.5% Tween 80. In thissituation there is formation of droplet suspension which turns intosolid microbeads when the temperature is raised to 80-90° C. for threeto five hours. The resulting beads are dried and organic solventsextracted. They are then swollen in an aqueous solution of collagen inphosphate buffer at neutral pH. Embedded collagen is then crosslinkedwith glutaraldehyde as described in Examples 1 and 2. The resultingbeads possess cationic charges to interact with cell tissues andcollagen for cell adhesion, and a chemotactic agent for cell growth andbiocompatibility. They are suitable as tissue bulking agent.

Example 4 Preparation of Hydrophilic Silicone Beads for Cell Adhesionand Tissue bulkinq

[0102] 10 gram of silicone beads of a diameter of 20-300 μm aresuspended in 30 ml of a solution of hexadecylamine (10 mg/ml) inethylacetate. The suspension is stirred for two hours and 100 ml ofethanol is added. A 1 M ammonium sulfate or sodium chloride solution inwater is added slowly until a 300 ml suspension is obtained. Theamino-containing silicone beads are then reacted with abutanedioldiglycydylether in alkaline conditions. Epoxy derivatives arethus obtained on which gelatin is coupled using a method well known inthe art. The resulting beads have the target properties ofbiocompatibility, hydrophilicity, non-biodegradability and cell adhesionby the presence of cationic amino groups and of gelatin as a cell growthpromoting agent. They are suitable for tissue bulking in accordance withthe present invention.

Example 5 Preparation of Beads for Tissue Bulking Containing AdhesionFactors

[0103] Beads prepared according to Example 2 were chemically activatedwith well known reagents used in the preparation of affinitychromatography sorbents. Activated beads were then used for theimmobilization of cell adhesion agents such as fibronectin orvitronectin or laminin. Adhesion agents were dissolved at 1-10 mg/ml ina coupling buffer (100 mM carbonate or borate buffer pH 8 to 10) and thesolution was mixed with the activated beads. The resulting beads possessthe target properties of cell adhesion and growth, non-biodegradabilityand were non-resorbable. They are suitable for cell adhesion andpermanent tissue bulking in accordance with the present invention.Similarly, beads prepared according to Examples 3 and 4 can also beused.

Example 6 Preparation of Spherical Polyacrylic Hydrogel Particles withChemotactic Properties

[0104] Microbeads commercially available under the name SPEC-70(BioSepra Inc., Marlborough, Mass.) are polyacrylic polyanionic beadswith elastic properties suitable for tissue bulking applications.However, these microbeads are not chemotactic and do not possesscationic charges. SPEC-70 microbeads are first drained under vacuum toeliminate water and then suspended in an aqueous solution of 1%chondroitin sulfate sodium salt in physiological conditions. Once thiscompound is absorbed on the bead structure, the beads are drained undervacuum and suspended in an aqueous solution containing 20% polylysine byweight. The suspension is shaken for a few hours and then drained undervacuum and rapidly washed with distilled water. The beads are thensuspended in a solution of 5% butanedioldiglycidylether in ethanol andshaken overnight. Under these conditions, the polylysine is crosslinkedas well as chondroitin sulfate. The resulting modified beads possessproperties such as cationic charge for cell adhesion and promotingagents for cell growth such as polylysine and chondroitin sulfate.

Example 7 Preparation of Radiopaque Microbeads with ChemotacticProperties for Tissue Bulking

[0105] Microbeads from Examples 2 were drained under vacuum and thensuspended in a saturated solution of barium chloride. They were shakenfor two hours at room temperature and then drained under vacuum toeliminate the excess of barium chloride solution. The beads weresuspended in a saturated solution of ammonium sulfate and shaken for twoadditional hours before elimination of the excess ammonium sulfate byvacuum filtration. This operation of contact with barium salts andammonium sulfate can be repeated several times until the resultingradiopaque precipitate inside the beads reaches the desired amount.Resulting beads have radiopaque properties without having lost theirinitial desirable properties for tissue bulking. The microbeads fromExamples 3, 4 and 6 can be similarly used.

Example 8 Preparation of Radiopaque Microbeads with ChemotacticProperties for Tissue Bulking

[0106] Microbeads from Example 6 coated with polylysine are washedextensively with distilled water and suspended in a solution of sodiumtriazoate. The suspension pH is adjusted at about 7 by addition ofacetic acid and shaken for several hours. The triazoate which is aradiopaque molecule is adsorbed tightly to the beads and the remainingreagents are eliminated by washing under vacuum. The resulting beadsstill possess cell promotion properties and now radiopacity as well.

Example 9 Introduction of Anti-inflammatory Drugs Inside the BulkingBeads

[0107] Microbeads described in the previous Examples may generate localtemporary inflammatory reactions when injected in the target tissue. Toavoid or decrease this phenomenon, the microbeads once coated withautologous cells can be filled with one or more anti-inflammatory drugs.The microbeads are cationic by their nature and can absorb anionic drugsby ion exchange effect.

[0108] Prior to injection microbeads are mixed with a 10 mg/mlanti-inflammatory anionic drug solution in sterile physiological saline.The suspension is shaken for several hours, and the beads filled withthe drug are recovered by filtration or centrifugation. The resultinganti-inflammatory containing microbeads may then be used as tissuebulking agents for use in the present invention.

Example 10 In Vitro Pre-adipocytes Adhesion and Growth on PolymericBeads

[0109] In order to assess the ability of polymeric beads from Example 2to allow adhesion and growth of pre-adipocytes, fresh pre-adipocyteswere collected and isolated from Wistar rat peri-epididymal fat tissue.Pre-adipocytes were then cultured in the presence of above describedmicrobeads at a concentration of about 7.1×10⁵ to about 1.7×10⁶ cells/mlusing the classical protocol for microcarrier culture in vitro. In afirst phase the cells adhere on the bead surface and then they grow tototally cover the bead surface. The total colonization period is about72 hours.

[0110] Pre-adipocytes from this type of culture show good growth andspecific biological activity associated with differentiation intoadipocytes (accumulation of lipids). Moreover these cells show thepresence of specific enzymatic markers such asglycerol-3-phosphate-dehydrogenase and malate dehydrogenase. Microbeadshaving cells adhered thereto are useful for tissue bulking for use inthe present invention. The polymeric beads of Examples 2 to 5 can besimilarly assessed.

Example 11 Culture of Pre-adipocytes and Myocytes on Microbeads in Vitroto Check Their Ability of Integrate into an in Vivo Tissue

[0111] Preadipocytes and smooth muscle cells were isolated from Wistarrats according to a classical protocol to eliminate most of othercontaminating cells. Separately these cells were cultured in a Petridish in the presence of Dulbecco's Modified Eagle Medium supplementedwith 10% fetal bovine serum. Gelatin-coated cationic microbeads preparedin accordance with Example 2 were added to cells cultured in vitro untilthey covered the surface of the Petri dish. Initial cell seedconcentration was 0.7×10⁶ cells/ml.

[0112] Repeated observations showed that cells adhered on the surface ofmicrobeads and further multiplied to cover all the surface of the beads.After 5 to 7 days of culturing, there was formation of a solid networkof beads where cells acted as a binder to consolidate the blocks ofseveral beads. In most cases there were formation of solid nondissociable aggregates comprising beads and cells.

[0113] When, after a growing period (generally 5 to 7 days), adifferentiating element such as 3,3′,5-triiodo-D-thyronine was added topreadipocytes, the preadipocytes started to accumulate fats asmicro-droplets within the cytoplasm.

[0114] Specific staining with 3,3′-dioctadecyloxacarbocyanineperchlorate or2′-[4-hydroxyphenyl]-5-[4-methyl-1-piperazinyl]2,5′-bi-1H-benzimidazoledemonstrated good adhesion of the cells on the bead substrate.

[0115] Staining of the cells with red oil at the beginning of thedifferentiating phase evidenced the accumulation of fats inside thecells.

[0116] In addition, specific enzymatic reactions of malic enzymeindicated that, at the end of the culture, resulting adipocytes werefunctionally viable with their major expressed characteristics. Thisenzyme is not expressed at the beginning of the culture and appearedsimultaneously with the accumulation of fats.

[0117] Smooth muscle cells were also followed in their proliferation byDNA synthesis assay; their adhesion on the substrate was followed as perpreadipocyte cells. Myocytes also showed good proliferation as well asadhesion on the beads.

Example 12 In Vitro Myocyte Adhesion and Growth on Polymeric Beads

[0118] In order to assess the ability of polymeric beads from Example 2to allow adhesion and growth of muscle cells, fresh smooth cell myocyteswere collected from rat esophagus according to classical procedures.Cells were then cultured in the presence of above described microbeadsat a concentration of about 10⁶ cells/ml using the classical protocolfor microcarrier culture in vitro. In a first phase the cells adhered onthe bead surface and then they grow until they cover the total beadsurface. The total colonization period was about 72 hours.

[0119] Myocytes from this type of culture showed good growth andbehavior and displayed the specific myosin marker. These microbeadshaving cells adhered thereto are useful for tissue bulking in accordancewith the present invention. The beads from Examples 2 to 5 can besimilarly assessed.

Example 13 Preparation of Injectable Suspension of Cell-microbeadParticles for in Vivo Bulking

[0120] At the issue of cell culture phase, the cell-bead particles arecollected by filtration and washed extensively with blood serum from thehost where the material is to be implanted. This operation ensures theelimination of foreign material from cell culture. The microbeads arethen suspended in a few ml of autologous serum (a ratio of beads/serumis about 1:1) and are ready to be injected within the tissue to bebulked by means of an appropriate syringe or other injection device.

Example 14 Preparation of Injectable Suspension of Cell-microbeadsParticles for in Vivo Bulking

[0121] Microbeads described in Example 2 are colonized with rat musclecells according to Example 10 and conditioned according to Example 13using rat serum diluted with physiological saline (50%-50%). The finalsterile suspension of cells anchored on beads (50% of volume isconstituted of beads and 50% of physiological saline) is injected in theright thigh muscle of a rat. Three months after bead injection themuscle was observed in its shape and histologically examined. Musclevolume should be larger than the left thigh muscle upon autopsy. Beadsinside the muscle mass should appear surrounded by fibroblastic cellswith no specific adverse inflammatory or necrosis effects.

[0122] The embodiments of the present invention described above areintended to be merely exemplary and those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, numerous equivalents to the specific proceduresdescribed herein. All such equivalents are considered to be within thescope of the present invention and are covered by the following claims.

[0123] The contents of all references described herein are herebyincorporated by reference.

[0124] Other embodiments are within the following claims.

What is claimed is:
 1. A method for treating gastroesophageal refluxdisease, which comprises administering to a mammal in need of suchtreatment a therapeutically effective tissue bulking amount ofbiocompatible hydrophilic microparticles, said administration being intothe lower esophageal sphincter or the diaphragm.
 2. The method of claim1, wherein the microparticles are cationic.
 3. The method of claim 1,wherein the microparticles comprise a positive charge on their surface.4. The method of claim 1, wherein said mammal is a human.
 5. The methodof claim 1, wherein the microparticles are pre-treated with,administered with, or coated with autologous cells.
 6. The method ofclaim 5, wherein the microparticles or cell coated microparticles arewashed with serum or whole blood prior to administration.
 7. The methodof claim 5, wherein the autologous cells are mucosal cells, musclecells, fat cells, or combinations thereof.
 8. The method of claim 1,wherein the microparticles are coated with or linked to at least onecollagen or a derivative thereof, glucosaminoglycans, or a mixturethereof.
 9. The method of claim 1, wherein the microparticles areadministered in a sterile and pyrogen-free injectable solution.
 10. Themethod of claim 1, wherein the microparticles are spherical.
 11. Themethod of claim 10, wherein the microparticles comprise a hydrophiliccopolymer which comprises in copolymerized form about 25 to about 99% byweight of neutral hydrophilic acrylic monomer, about 2 to about 50% byweight of one or more monomers having a cationic charge, and about 1 toabout 30% by weight of a functionalized monomer.
 12. The method of claim10, wherein said microparticles have diameters ranging from about 10 μmto about 1000 μm.
 13. The method of claim 1, wherein said administrationis made via syringe, catheter, or combinations thereof.
 14. The methodof claim 1, wherein said microparticles comprise or are administeredwith one or more of a therapeutic agent, an anti-inflammatory agent, anangiogenesis inhibitor, a radio active element, and an antimitoticagent.
 15. The method of claim 1, wherein the microparticles furthercomprise a cell adhesion promoter.
 16. The method of claim 15, whereinsaid cell adhesion promoter is selected from the group consisting offibronectin, laminin, chondronectin, entacin, epibolin, liver celladhesion molecule, serum spreading factor, collagen, heparin sulfates,dermatan sulfates, chonodroctin sulfates, glucosaminoglycans, andmixtures thereof.
 17. A sterile injectable solution suitable fortreating gastroesophageal reflux disease, which comprises: (a)biocompatible hydrophilic cationic microspheres, having a diameter of 10to 1000 μm, said microspheres comprising a neutral hydrophilic monomer,one or more cationic monomers, one or more functionalized monomers; and(b) autologous cells.
 18. The solution of claim 17, wherein themicrospheres further comprise a cell adhesion promoter.
 19. A method fortreating gastroesophageal reflux disease, which comprises: (a) preparingcationic microparticles which comprise biocompatible and hydrophilicpolymers; (b) administering the resulting microparticles to a mammal byinjection into walls of a sphincter located where the esophagus meetsthe stomach.
 20. The method of claim 19, wherein the microparticlesfurther comprise a cell adhesion promoter.